Field of the Invention and Related Art Statements
The present invention relates to method and apparatus for measuring an immunological antigen-antibody reaction with the aid of phase information of polarized light scattered by fine particles suspended in a reaction liquid.
There has been developed an immunological analysis for measuring immune substances, hormones, medicines, and various components such as immune regulators faintly contained in living bodies by utilizing specific immunological reactions. The immunological analysis may be roughly classified into a labeling immunological analysis in which enzymes and isotopes are used as an indicator substance, and a non-labeling immunological analysis in which antigen-antibody complexes are directly measured.
In the former labeling immunological analysis, there have been widely known radio immuno assay (RIA), enzyme immuno assay (EIA) and fluorescent immuno assay (FIA). These assays have an advantage that a high sensitivity can be attained, but also have a drawback that handling of isotopes and wasted liquid is difficult, measuring periods are long and labeling reagents are expensive so that the test cost per sample, i.e. running cost, is liable to be high.
In non-labeling immunological analysis, there have been developed immuno electrophoresis, immuno diffusion and sedimentation. These methods are rather simple, but do not have sufficiently high sensitivity, quantitativeness and reproducibility necessary for precise measurement.
In "Immuno chemistry", vol. 12, No. 4 (1975) pages 349 to 351, there has been proposed an immunological analysis in which an antigen or antibody bound on surfaces of fine particles are reacted with an antibody or antigen contained in a test liquid, and average diffusion constant which is an indicia of the Brownian motion of aggregates composed of agglutinated particles is measured from a variation in a spectral width of laser light scattered from a solution of particles. This method has a merit that no reagent is depleted. However, since the spread of the spectrum due to the Doppler effect owing to the Brownian motion of aggregates is detected by a spectrometer, the apparatus is liable to be large in size and expensive in cost. Further, error might be induced when the spectrometer is driven mechanically, so that precision and reproducibility become worse. Moreover, in this known method, because the average diffusion constant is measured from the spectral width, the amount of available information about the antigen-antibody reaction is limited.
In order to avoid the above mentioned drawbacks, the inventor of the present invention has conducted various experiments and analyses and proposed a novel method of measuring the immunological reaction by detecting a fluctuation in intensity of light scattered by particles. Such a method has been disclosed in U.S. patent application Ser. No. 754,272 filed on July 12, 1985.
After developing such a method, the inventor has further conducted various experiments and has designed an improved method in which linearly polarized light is made incident upon a reaction liquid via a polarizer and light scattered by particles is detected through an analyzer which has a polarization plane perpendicular to that of the polarizer. This method is based on the fact that when the linearly polarized light is scattered by the particles, the polarization condition of the scattered light has an intimate relation with the antigen-antibody reaction.
The inventor has experimentally confirmed that the above method has the following drawbacks. In order to improve the measuring sensitivity when a concentration of particles is increased, there occurs multiple scattering and this results in the scattered light containing a polarization component perpendicular to the polarization plane of the incident linearly polarized light although the antigen-antibody reaction does not take place in the cell, so that the sensitivity might be decreased and the immunological reaction can not be measured accurately.